Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids

toxins Article Activation and Desensitization of Peripheral Muscle and Neuronal Nicotinic Acetylcholine Receptors by Selected, Naturally-Occurring Pyridine Alkaloids Benedict T. Green 1,*, Stephen T. Lee 1, Kevin D. Welch 1, Daniel Cook 1 and William R. Kem 2 1 Poisonous Plant Research Laboratory, Agricultural Research Service, United States Department of Agriculture, 1150 E. 1400 N., Logan, UT 84341, USA; [email protected] (S.T.L.); [email protected] (K.D.W.); [email protected] (D.C.) 2 Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, 1200 Newell Road, Gainesville, FL 32610-0267, USA; wrkem@ufl.edu * Correspondence: [email protected]; Tel.: +1-435-752-2941 Academic Editor: Peter S. Spencer Received: 29 January 2016; Accepted: 24 June 2016; Published: 4 July 2016 Abstract: Teratogenic alkaloids can cause developmental defects due to the inhibition of fetal movement that results from desensitization of fetal muscle-type nicotinic acetylcholine receptors (nAChRs). We investigated the ability of two known teratogens, the piperidinyl-pyridine anabasine and its 1,2-dehydropiperidinyl analog anabaseine, to activate and desensitize peripheral nAChRs expressed in TE-671 and SH-SY5Y cells. Activation-concentration response curves for each alkaloid were obtained in the same multi-well plate. To measure rapid desensitization, cells were first exposed to five potentially-desensitizing concentrations of each alkaloid in log10 molar increments from 10 nM to 100 µM and then to a fixed concentration of acetylcholine (ACh), which alone produces near-maximal activation. The fifty percent desensitization concentration (DC50) was calculated from the alkaloid concentration-ACh response curve. Agonist fast desensitization potency was predicted by the agonist potency measured in the initial response. Anabaseine was a more potent desensitizer than anabasine. Relative to anabaseine, nicotine was more potent to autonomic nAChRs, but less potent to the fetal neuromuscular nAChRs. Our experiments have demonstrated that anabaseine is more effective at desensitizing fetal muscle-type nAChRs than anabasine or nicotine and, thus, it is predicted to be more teratogenic. Keywords: anabaseine; anabasine; epibatidine; nicotinic acetylcholine receptor; nicotine; pyridine alkaloid 1. Introduction In grazing livestock, the in utero exposure to certain piperidinyl-pyridine alkaloid plant toxins from Lupinus, Conium, and Nicotiana spp. can cause developmental defects (terata) by inhibiting fetal movement [1]. These defects (arthrogyrposis, kyposis, lordosis, scoliosis, torticollis, and cleft palate) are termed multiple congenital contracture-type (MCC-type) deformities [2]. The mechanism by which these plant toxins inhibit fetal movement has been postulated to involve activation, and then desensitization, of fetal muscle-type nicotinic acetylcholine receptors (nAChRs) that are expressed by the developing fetus [1]. This mechanism is similar to the mechanism of neuromuscular block caused by depolarizing muscle relaxants, like succinylcholine [3]. These alkaloids from Lupinus, Conium, and Nicotiana spp. cause crooked calf syndrome in extensively-grazed cattle in the Western United States. Crooked calf syndrome negatively impacts the welfare of the calf and creates an economic burden due to increased management and veterinary costs. Toxins 2016, 8, 204; doi:10.3390/toxins8070204 www.mdpi.com/journal/toxins Toxins 2016, 8, 204 2 of 12 Toxins 2016, 8, 204 2 of 12 NicotinicNicotinic acetylcholine acetylcholine receptors receptors are are ligand-gated ligand‐gated cation channels channels and and members members of of the the cys cys-loop‐loop receptorreceptor family. family. Each Each receptor receptor is ais pentameric a pentameric complex complex containing containing five five homologous homologous subunits. subunits. There There are 17are distinct 17 distinct nAChR nAChR subunits; subunits;α1–10 α,1–10β1–4, β,1–4γ,, γδ,, δ and, and" ε [4[4,5],5] that that areare expressed in in vertebrates. vertebrates. More More than than tenten different different subunit subunit combinations combinations have have been been reported reported to occur in in mammals. mammals. In In each each receptor receptor at atleast least twotwo C-loop-containing C‐loop‐containingα α subunitssubunits are are present;present; theythey provide most most of of the the two two acetylcholine acetylcholine binding binding sitessites that that must must be be occupied occupied by by agonists agonists for for maximalmaximal activation.activation. All All nAChRs nAChRs can can exist exist in in multiple multiple conformationconformation states, states, including including resting resting (closed (closed channel),channel), activated (open (open channel), channel), and and desensitized desensitized (agonist(agonist occupied occupied but but closed closed channel) channel) states, states, all of all which of which display display different different allosterically-linked allosterically‐ affinitieslinked affinities for agonists and antagonists [6,7]. Transitions between the various receptor states depend, for agonists and antagonists [6,7]. Transitions between the various receptor states depend, to some to some degree, upon the chemical structure of the agonist. For example, the excitatory degree, upon the chemical structure of the agonist. For example, the excitatory neurotransmitter neurotransmitter acetylcholine (ACh) is more potent at activating fetal muscle‐type nAChR acetylcholine (ACh) is more potent at activating fetal muscle-type nAChR expressed by TE-671 cells expressed by TE‐671 cells than the pyridine alkaloid nicotine, with EC50 values 0.3 and 17.6 μM, than the pyridine alkaloid nicotine, with EC values 0.3 and 17.6 µM, respectively [8]. Prolonged respectively [8]. Prolonged exposure of TE50‐671 cells to ACh also produces a more profound exposuredesensitization of TE-671 than cells nicotine to ACh [9]. also Differences produces in aagonist more profoundactions at desensitizationthe muscle receptor than have nicotine been [ 9]. Differencespartially explained in agonist by actions differences at the in muscle the types receptor of non‐covalent have been bonding partially interactions explained between by differences agonists in theand types the of different non-covalent nAChRs bonding that are interactions related to differences between agonists in the chemical and the structures different nAChRsof the agonists that are related(Figure to differences1A) [10,11]. in the chemical structures of the agonists (Figure1A) [10,11]. Figure 1. Chemical structures of compounds tested in this research (A). Illustration of experimental Figure 1. Chemical structures of compounds tested in this research (A). Illustration of experimental design (B), depicting compound additions one (measuring agonist potency and causing design (B), depicting compound additions one (measuring agonist potency and causing desensitization) desensitization) and two (measuring desensitization), and the KCl addition that served as a and two (measuring desensitization), and the KCl addition that served as a depolarizing calibrant. depolarizing calibrant. The second compound addition was ACh at 10 μM (SH‐SY5Y cells) or 1 μM The second compound addition was ACh at 10 µM (SH-SY5Y cells) or 1 µM (TE-671 cells).The change (TE‐671 cells).The change in relative fluorescence of the dye‐loaded cells was detected using a in relative fluorescence of the dye-loaded cells was detected using a Flexstation III multi-mode Flexstation III multi‐mode microplate reader. microplate reader. In this research, we investigated four nAChR agonists (epibatidine, the most potent known nAChRIn this agonist, research, nicotine, we investigated anabasine, and four anabaseine) nAChR agonists for their (epibatidine, ability to activate the most and potentthen rapidly known nAChRdesensitize agonist, nAChRs. nicotine, The anabasine, rapid type and of desensitization anabaseine) for examined their ability in this to activatestudy has and been then termed rapidly desensitize“classical desensitization” nAChRs. The rapid and develops type of on desensitization a second time scale examined at the agonist in this studyconcentrations has been used termed in “classical desensitization” and develops on a second time scale at the agonist concentrations used in Toxins 2016, 8, 204 3 of 12 Toxins 2016, 8, 204 3 of 12 our investigation [7]. We chose to examine classical desensitization based on our previously reported researchour investigation of plant toxin-induced [7]. We chose inhibition to examine of fetalclassical movement desensitization in goats, based which on has our documented previously reported the nearly immediateresearch abolitionof plant toxin of fetal‐induced goat inhibition movement of by fetal several movement pyridine in goats, alkaloids, which but has not documented nicotine [12 the,13 ]. In thisnearly study immediate we used abolition two cell linesof fetal with goat fetal movement characteristics by several as models pyridine to study alkaloids, receptor but desensitization not nicotine of the[12,13]. two In peripheral this study nAChRs: we used TE-671two cell cells lines expressing with fetal fetalcharacteristics human muscle-type as models to nAChR study (receptorα12β1γδ ) anddesensitization SH-SY5Y cells of derivedthe two peripheral from embryonic nAChRs: central TE‐671 nervous cells expressing system tissuefetal human expressing muscle markers‐type characteristicnAChR (α1 of2β1 immatureγδ) and

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